Multiple hybrid immunoassay

ABSTRACT

The invention relates to compositions and methods for the immunoassay of an analyte of interest. The analyte is detected in an immunoassay using three or more antibodies, wherein each antibody specifically binds to a different epitope on the analyte. When the analyte of interest in a clinical marker for an acute disease, the detection of the analyte by immunoassay is a diagnosis of the occurrence of the disease.

FIELD OF THE INVENTION

[0001] The present invention relates to the diagnosis of diseases andmedical events, notably myocardial infarction (“MI”). More specifically,the present invention relates to the detection of a clinical marker of adisease or medical event, in particular MI, using multiple antibodies,each antibody having a different specificity for the clinical marker.The present invention also relates to reagents and apparatuses used inthe diagnosis of a disease or medical event, in particular MI.

BACKGROUND

[0002] Diagnosis of acute disease is often based on immunoassay, e.g.enzyme-linked immunosorbent assay (“ELISA”), detection of abnormallevels of clinical markers, such as proteins, enzymes and hormones inbiological fluids, particularly when the concentration changes quicklyduring the acute phase of disease. ELISA systems allowing for the rapidand simple diagnosis of the occurrence of an acute disease, such as theoccurrence of an MI, are therefore extremely important.

[0003] In the past, clinical markers for diagnosing the occurrence of anMI included lactate dehydrogenase (“LDH”) and glutamic oxaloacetictransaminase (“GOT”), though these were not very specific. Problems withLDH and GDH led to the use of the MB isoenzyme of creatine kinase(“CK-MB”) as a clinical marker for the diagnosis of MI. However, CK-MBcan also be found in skeletal muscle and in blood after skeletal muscleinjury. Thus, CK-MB is not completely specific for cardiac muscle.Another disadvantage of CK-MB as a clinical marker of MI is that thelevel of CK-MB in the skeletal muscle varies with the degree of skeletalmuscle regeneration, information which may often not be known whenadministering a test or analyzing a test result for MI. Anotherdisadvantage of testing for CK-MB is that CK-MB levels remain elevatedfor only 2-3 days after the onset of chest pain. For patients admittedafter that time, the CK-MB test will be of limited value. Thus, due tothe lack of specificity when assaying CK-MB, and the limited time framefor its use as a diagnostic tool, CK-MB is not an ideal clinical markerfor diagnosing MI.

[0004] Cardiac troponin I (“cTnI”) is now used as an accuratecardiac-specific biological parameter detectable in serum very soonafter MI and remaining present for more than 2-3 days after the onset ofMI. Troponin is present in cardiac tissue as a complex of threesubunits: Troponin T (“TnT”), the tropomyosin binding subunit, TroponinC (“TnC”), the Ca²⁺ binding subunit; and Troponin I (“TnI”), thesub-unit which inhibits the actomyosin Mg²⁺ ATPase. TnI is a thinfilament regulatory protein complex, which confers calcium sensitivityto the cardiac and striated muscle.

[0005] Human Troponin I exists in three isoforms: two skeletal muscleisoforms (fast and slow) (MW=19.8 kDa) and a cardiac TnI isoform(“cTnI”) with an additional 31 residues on the N-terminus resulting in amolecular weight of 23 kDa (209 amino acids). Cardiac TnI is uniquelylocated in the myocardium where it is the only isoform present. CardiacTnI rapidly appears in human serum (within approximately 4 to 6 hours)following a MI. It reaches a peak level after 18-24 hours and remains atelevated levels in the blood stream for up to 6-10 days. As a result,cTnT released into the circulation from the myocardium is very specificfor myocardial injury.

[0006] Elevated cTnI levels in blood may be used to diagnose MI anddistinguish other heart related events and diseases. Immunoassay systemscapable of accurately detecting human cTnI would be valuable to themedical community for diagnosing the occurrence of MI. For moreinformation on the utility of cTnI testing, see Apple and Wu, Myocardialinfarction redefined: Role of cardiac troponin testing. ClinicalChemistry 47, 377-9, (2001), which is hereby incorporated by reference.

[0007] Cardiac TnI exists in multiple subforms in blood as a result ofmodifications such as proteolytic cleavage, phosphorylation, chemicaloxidation, chemical reduction, cleavage of amino acid residues, andchemical modification of amino acid moieties. For example, amino acids 1to 25 and 150 to 209 are generally not found on cTnI subforms in serumas a result of proteolysis. The many different subforms of cTnIcirculating in the bloodstream are predominantly found in complexes withother proteins. See Katrukha (1997). For example, cTnI may be complexedwith cTnT and cTnC (“cITC”).

[0008] In many instances, the chemical modifications and complexation ofcTnI in the bloodstream eliminate or block the binding sites for ELISAreagents on some subforms of cTnI, thereby making the epitopes of theELISA reagents unavailable for binding. For more information on cTnIepitopes and cTnI instability, see Morjana et al., Degradation of Humancardiac troponin I after myocardial infarction: Biotechnol. Appl.Biochem. (1998), 28, 105-111; Gaelle Ferrieres et al. Human cardiactroponin I: precise identification of antigenic epitopes and predictionof secondary structure: Clin. Chem. (1998), 44, 487-493; Katrukha etal., Troponin I is released in bloodstream of patients with acutemyocardial infarction not in free form but as complex: Clin. Chem.(1997), 43, 1379-1385; and Katrukha et al., Degradation of cardiactroponin I: implication for reliable immunodetection: Clin. Chem.(1998), 44, 2433-244, which are incorporated herein by reference.

[0009] For most commercially important clinical marker analytes, anumber of efficient monoclonal antibodies have been developed which binda particular epitope of an analyte. The difference between antibodiesare the specific location of binding to the analyte, affinity for theanalyte, and cross-reactivity with other potential interferents withinthe sample. ELISA assays have been traditionally developed by pairwisetesting of available antibodies for use either as the capture reagent orsignal reagent.

[0010] In the development of ELISA assays for cTnI, much effort has beenexpended in optimizing the antibody and enzyme reagents used. However,the likelihood of epitopes being unavailable for binding on the manysubforms of cTnI present in serum and complexation with other proteinsmakes detection of cTnI significantly difficult and typically causecurrent ELISA reagents to underestimate actual cTnI levels in a sample.Furthermore, the many different subforms of cTnI with different epitopesavailable for binding cause current ELISA assays to give significantlydifferent results from sample to sample, from reagent set to reagentset, and as a function of time.

[0011] Thus, there exists a need to develop methods of detecting aclinical marker and subforms thereof, in particular cTnI, for thediagnosis of an acute disease event, in particular MI. The presentinvention satisfies this need and provides reagents, kits, andapparatuses for detecting a clinical marker and subforms thereof, inparticular cTnI.

SUMMARY

[0012] The present invention is related to the detection of an analyteof interest. The analyte of interest may be cTnI. In an exemplaryembodiment of the present invention, an immunoassay composition fordetecting an analyte of interest may comprise three or more antibodies,wherein each antibody is capable of binding to a different epitope onthe analyte. At least two of the three different antibodies which bindto the analyte are also capable of binding to at least two differentepitopes on a subform of the analyte. At least one of the epitopes onthe analyte of interest is unavailable for binding on the subform.

[0013] In another exemplary embodiment of the present invention, animmunoassay device for detecting an analyte of interest may comprise twoor more antibodies, wherein each antibody binds to a different epitopeon the analyte and wherein the two or more antibodies are bound to atleast one surface. At least one of the two different antibodies whichbind to the analyte is also capable of binding to at least one differentepitope on a subform of the analyte. At least one of the epitopes on theanalyte of interest is unavailable for binding on the subform.

[0014] In another exemplary embodiment of the present invention, animmunoassay kit for detecting an analyte of interest may comprise threeor more antibodies, wherein each antibody is capable of binding to adifferent epitope on the analyte. At least two of the three differentantibodies which bind to the analyte are also capable of binding to atleast two different epitopes on a subform of the analyte. At least oneof the epitopes on the analyte of interest is unavailable for binding onthe subform.

[0015] In another exemplary embodiment of the present invention, asandwich immunoassay product may comprise an analyte of interest and asubform of the analyte. At least three different epitopes on the analyteare available for binding by at least three different antibodies. Atleast three different antibodies are bound to different epitopes on theanalyte. At least two of the three epitopes on the analyte are availablefor binding on the subform. At least two of the at least three antibodieare bound to different epitopes on the subform. At least one of theepitopes on the analyte of interest is unavailable for binding on thesubform.

[0016] In another exemplary embodiment of the present invention, apatient may be diagnosed for the occurrence of an acute disease, such asmyocardial infarction, by applying a sample obtained from the patient toa surface comprising at least two antibodies which bind to differentepitopes on cTnI, wherein at least one of the two antibodies is capableof binding to a different epitope on a subform of cTnI, and wherein atleast one epitope of the at least two antibodies is unavailable forbinding on the subform. A third antibody is then added which binds toyet another epitope on cTnI and the subform. The extent of binding ofthe third antibody to cTnI and the subform is then measured.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 shows the standard sandwich assay for detecting an analyteof interest, wherein a capture antibody (Ab₁) and signal antibody (Ab₂)are directed to a first and second epitope (e₁ and e₂), respectively, ofan analyte of interest (An). The signal antibody is labeled with anenzyme (Enz), which is used for signal generation by convertingsubstrate (S) to product (P).

[0018]FIG. 2 is a representation of an analyte of interest (An) withthree epitopes (e₁, e₂ and e₃). As the analyte is modified, formscomplexes or adopts alternative conformations, a subform is producedwith one or more of the epitopes unavailable for binding by antibodies.Each epitope that is unavailable for binding is identified by a circle,whereas each epitope that is available for binding is identified withouta circle. Each subform may be further modified so that additionalepitopes are unavailable for binding by antibodies.

[0019]FIG. 3 shows a modified sandwich assay for detecting an analyte ofinterest (An) and subforms thereof using one capture antibody (Ab₁) andmore than one signal antibody (Ab₂ and Ab₃). The assay is able to detectthe presence of the analyte and all subforms thereof that have at leastone epitope for the capture antibody (e₁) and at least one epitope for asignal antibody (e₂ or e₃). All epitopes for binding are shown bound byan antibody; however, the analyte or subforms thereof need to be boundby only one capture antibody and one signal antibody to be detected.

[0020]FIG. 4 shows a modified sandwich assay for detecting an analyte ofinterest (An) and subforms thereof using more than one capture antibody(Ab₁ and Ab₂) and one signal antibody (Ab₃). The assay is able to detectthe presence of the analyte and all subforms thereof that have at leastone epitope for the signal antibody (e₃) and at least one epitope for acapture antibody (e₁ or e₂). All epitopes for binding are shown bound byan antibody; however, the analyte or subforms thereof need to be boundby only one capture antibody and one signal antibody to be detected.

[0021]FIG. 5 shows a modified sandwich assay for detecting an analyte ofinterest (An) using more than one capture antibody (Ab₁ and Ab₂) andmore than one signal antibody (Ab₃ and Ab₄). The assay is able to detectthe presence of the analyte and all subforms thereof that have at leastone epitope for a capture antibody (e₁ or e₂) and at least one epitopefor a signal antibody (e₃ or e₄). All epitopes for binding are shownbound by an antibody; however, the analyte or subforms thereof need tobe bound by only one capture antibody and one signal antibody to bedetected.

[0022]FIG. 6 shows one antibody (Ab₁) or more than one antibody (Ab₁ andAb₂) binding to a different epitope (e₁ or e₂) on the analyte ofinterest (An) and conjugated to a common member, wherein the commonmember is a surface (FIG. 6A), microparticle (FIG. 6B), and apolypeptide, such as an enzyme (FIG. 6C).

[0023]FIG. 7 shows a mixture of common members individually conjugatedwith a different antibody (Ab₁ or Ab₂), each antibody binding to adifferent epitope (e₁ or e₂) on the analyte being assayed (An), whereinthe common member is a microparticle (FIG. 7A) or polypeptide, such asan enzyme (FIG. 7B).

[0024]FIG. 8 shows a common member, such as a microparticle, conjugatedto two or more antibodies (Ab₁ and Ab₂), conjugated to another commonmember, such as a surface, wherein two or more capture antibodies areeither (i) individually conjugated to different microparticles (FIG.8A), or (ii) conjugated together on a microparticle (FIG. 8B). [00251FIG. 9 depicts the primary structure of subform of cardiac troponin I(cTnI) without amino acids 1 to 25 and 150 to 209, and shows thelocation for epitopes of the cTnI antibodies.

[0025]FIG. 10 shows the amperometric signals from different single-useassays prepared with a first capture antibody (CB1), a second captureantibody (CB2), or a combination of first and second capture antibodies(CB12) to a whole blood sample spiked with free cTnI and two levels ofcITC complex.

[0026]FIG. 11 shows a plot of the average signal obtained from a wholeblood sample spiked with 6.0 ng/mL cITC complex immediately afterpreparation and after one-day incubation at room temperature usingcartridges prepared with either CB1 or CB12 coated sensors and with asignal producing enzyme conjugated to a first antibody (EC1), a secondantibody (EC2) or a mixture of EC1 and EC2 (EC12).

[0027]FIG. 12 shows elution profiles for Fab-ALP conjugates withdifferent ratios of ALP to Fab.

DETAILED DESCRIPTION

[0028] 1. Definitions

[0029] To aid in the understanding of the present invention, severalterms are defined below.

[0030] “Analyte” means a biological or chemical substance that iscapable of being bound by at least three different antibodies, andincludes subforms thereof that may be bound by at least three differentantibodies.

[0031] “Antibody” means a polypeptide or derivative thereof thatspecifically binds to an epitope on an analyte of interest or subformthereof.

[0032] “Capture antibody” or “capture reagent” means an antibody thatspecifically binds to an analyte of interest or subform thereof, whereinsaid antibody is immobilized on a surface either (i) before, (ii) afteror (iii) during binding to said analyte or subform thereof.

[0033] “Detect”, “detected,” or “detectable,” when used to describe asignal of a signal-generating element, means a signal capable of beingdistinguished from background.

[0034] “Epitope” means a binding site for an antibody or other bindingmember on an analyte of interest or subform thereof. An epitope is“unavailable for binding” if the epitope is either not present orinaccessible for binding by an antibody.

[0035] “Immunoassay” or “sandwich immunoassay” means a method ofsimultaneous sandwich, forward sandwich and reverse sandwichimmunoassays, and includes competitive immunoassays thereof, all ofwhich are well understood by those skilled in the art.

[0036] “Subform,” when used to describe an analyte, means the product ofa chemical reaction, a member of a complex with other biological orchemical substances, alternative conformations, or combinations thereofthat may be bound by at least two different antibodies.

[0037] “Sensing element” means any device or apparatus that is capableof detecting a signal.

[0038] “Signal antibody” or “signal reagent” means an antibody thatspecifically binds to an analyte of interest or subform thereof, whereinsaid antibody is attached to a signal-generating element via a covalentlinkage, hydrophobic interactions, hydrophilic interaction, ionicinteractions, Van der Waal forces, or a combination thereof.

[0039] “Signal-generating element” means a biological, chemical, orradioactive substance that (i) produces a detectable signal, directly orindirectly, or (ii) is itself detectable.

[0040] “Surface” means a support that may be separated from a solution.

[0041] 2. Analyte

[0042] The present invention relates to the detection of an analyte andsubforms thereof. The analyte may be a clinical marker of a diseasestate from a patient believed to have suffered an acute disease event,wherein the clinical marker is also present in one or more subforms. Inan acute disease state which is amenable to analysis by this invention,the clinical marker may be a transiently elevated substance in the bloodwhich is released in a significant quantity at, after, or before thetime of the acute disease event of interest. The elevated concentrationof the clinical marker analyte decreases as endogenous conversionfactors act upon the clinical marker to produce subforms of the analyte.Subforms derived from the clinical marker may be transiently elevated ina serial manner, as each is first created then metabolized by endogenousconversion factors. The period of transient elevation may be for asshort as a few hours to as long as several weeks.

[0043] The analyte may be a biological substance such as a protein,glycoprotein, enzyme, etc., which is released in small quantities at theoccasion of an acute disease event such as a heart attack, stroke, or atthe occasion of a traumatic injury such as a broken bone or a hematoma.The analyte may be normally absent in such increased quantities and maybe converted to a one or more subforms over time by endogenousconversion factors including, but not limited to proteolytic cleavage,phosphorylation, chemical oxidation, chemical reduction, cleavage ofamino acid residues, and chemical modification of amino acid moieties.The analyte may be a protein including, but not limited to, TnI, TnT,CK-M, CK-B, myoglobin, hCG, TSH, FSH, pneumococcal PCA, apolipoproteins,C-reactive protein (CRP), brain natriuretic protein (BNP) and itspro-form pro-BNP, human leukocyte antigen and human apolipoproteins. Apreferred analyte is cTnI.

[0044] The analyte has at least three different epitopes that areavailable for binding by an antibody (FIG. 2). A subform of the analytehas at least two different epitopes that are available for binding by anantibody and the epitopes of the subform are also present on the analyte(FIG. 2). At least one epitope of the analyte is unavailable for bindingon the subform. An epitope of the analyte unavailable for binding on thesubform may be as a result of complexation or alternative conformations,as well as the endogenous conversion factors described above.

[0045] The analyte may be in a native or natural form. The analyte mayalso be a derivative of the native or natural form. For example, in thecase of cTnI the analyte may be full length cTnI monomer or a derivativethereof that is either free or part of cITC complex. In addition, theanalyte may be any form of cTnI that is released into serum inconnection with an MI. The analyte may also be any modified form of cTnIpresent in serum including, but not limited to, cTnI as shown in FIG. 9.The analyte may also be any modified form of cTnI that has one or moreepitopes that are unavailable for binding on any precursor of theanalyte. In order to be an analyte, the only requirement is that themodified or derivative form of cTnI must have at least three epitopesavailable for binding by three different antibodies.

[0046] The present invention may also be used to detect an analyte andsubforms thereof that are present at low concentration where highsensitivity is required.

[0047] 3. Immunoassay

[0048] Analytes have traditionally been detected using a system based onthe sandwich assay depicted in FIG. 1 wherein a first monoclonal orpolyclonal antibody for capture is attached to a surface, and a secondmonoclonal or polyclonal antibody is labeled with a signal-generatingelement (e.g., an enzyme). For more information on commercialimmunoassay products and the technology on which they are based, seeWild, (Ed), The Immunoassay Handbook, Stockton Press N.Y., 1994, whichis incorporated herein by reference. The traditional sandwich assayfails, however, to adequately detect analytes that undergo complexation,alternative conformations or modification that make epitopes for thecapture antibody, signal antibody, or both unavailable for binding.

[0049] The present invention relates to the use of a sandwichimmunoassay for detecting an analyte and one or more subforms thereof(FIG. 2). Subforms of the analyte may be due to causes includingmutations, complexation, chemical modification, proteolysis orrearrangement. The present invention may also be used to detect morethan one related analyte, such as an active protein and its inactiveprecursor or a class of similar analytes for which a single detectionassay is developed.

[0050] The analyte of interest and one or more subforms thereof may bedetected by performing an immunoassay using antibodies specific for morethan one epitope on the analyte either (i) on the capture side of thesandwich (FIG. 4), (ii) signal side of the sandwich (FIG. 3), (iii) orboth (FIG. 5). By targeting at least three epitopes, in total, on theanalyte of interest, the multiple sandwich assay of the presentinvention may detect the presence of the analyte and subforms thereofeven if certain epitopes are unavailable for binding on the subform, solong as there is at least one epitope capable of binding by a captureantibody and at least one epitope available for binding by a signalantibody. The present invention is therefore able to detect analytes andmultiple subforms thereof that appear within a sample.

[0051] One of skill in the art will recognize that the inventiondescribed herein may detect an analyte of interest by using any bindingmember that is capable of specifically binding to an epitope on theanalyte of interest. Binding members include, but are not limited to,extracellular or intracellular receptors, polynucleotides, peptidenucleic acids, and derivatives thereof.

[0052] The use of multiple antibodies in the practice of the presentinvention is contrasted with the use of polyclonal antibodies intraditional sandwich assays; in addition, the present invention issuperior. Within a given polyclonal preparation, the specific epitopesrecognized and the ratio of various antibodies in the preparation isgenerally unknown. Furthermore, the epitopes recognized and the ratio ofvarious antibodies vary between polyclonal preparations. Anotherdeficiency in the polyclonal approach is that the binding of individualantibodies within the polyclonal preparation will have diverse bindingaffinities. As a result, a significant portion of polyclonalpreparations are antibodies which have sub-standard analyticalperformances or which bind epitopes that compete with other antibodiesin the preparation. By contrast, the present invention allows thepreparation of controlled multi-epitopic reagents with more efficientassay characteristics because of better binding affinities and lessreagent required for a given assay signal.

[0053] In one exemplary embodiment of the invention, the immunoassay isbased on a first antibody attached to a surface (i.e., “captureantibody” or “capture reagent”) and at least a second and thirdantibody, wherein each antibody binds to a different epitope on theanalyte. The antibodies not attached to a surface may be labeled with asignal-generating element (i.e., “signal antibody” or “signal reagent”).See FIG. 3.

[0054] In another exemplary embodiment of the invention, the immunoassayis based on at least a first and second capture antibody and a thirdantibody. The antibody not attached to a surface may be a signalantibody. See FIG. 4.

[0055] In another exemplary embodiment of the invention, the immunoassayis based on at least a first and second capture antibody and at least athird and fourth antibody. The antibodies not attached to a surface maybe signal antibodies. See FIG. 5.

[0056] 4. Antibodies

[0057] The antibodies of the present invention may be any antibody thatspecifically binds to an epitope available for binding on an analyte ofinterest or a subform thereof. The antibodies of the present inventioninclude antibodies of classes IgG, IgM, IgA, IgD, and IgE, and fragmentsand derivatives thereof including Fab, F(ab′)₂ and single chainantibodies. The antibodies of the present invention include monoclonalantibodies, polyclonal antibodies, affinity purified antibodies, ormixtures thereof which exhibit sufficient binding specificity to anepitope of the analyte or subform thereof. Monoclonal antibodies andfragments and derivatives thereof are preferred in the practice of theinvention. The antibodies of the present invention preferably bind toepitopes of the analyte sufficiently removed from each other such thatthe antibodies do not mutually interfere with binding to the analyte orsubform thereof. Appropriate antibodies for an analyte of interest maybe chosen by means of mapping combinations of available antibodies toknown epitope sites on the analyte using methods known in the art.

[0058] Numerous methods exist for preparing antibody fragments,including the use of common enzymes such as pepsin, papain, ficin, andtrypsin. For more information on antibody preparation and antibodyfragmentation, see Harlow and Lane, Antibodies, A Laboratory Manual,Cold Spring Harbor Laboratory, N.Y., 1988, which is hereby incorporatedby reference.

[0059] The antibodies of the present invention may be specific for humancTnI. The antibodies may specifically recognize epitopes located on theprimary sequence of cTnI as indicated in FIG. 9. The antibodies may alsobe chosen from those antibodies indicated in FIG. 9.

[0060] 5. Capture

[0061] Capture antibodies of the present invention may be immobilized byconjugating one or more antibodies to a surface. See, e.g., FIG. 6A andFIG. 6B. A wide variety of compounds may be employed as the surface, theprimary consideration being the binding of the antibody to the surface,the absence of interference with the signal generating element, and theabsence of interference with the examination of the label. Inparticular, if a fluorescence or chromogenic spectrum is being measured,the surface should not provide interference.

[0062] Organic and inorganic polymers, both natural and synthetic, maybe employed as the surface. Examples of suitable polymers includepolyethylene, polypropylene, polybutylene, poly(4-methylbutylene), butylrubber and other synthetic rubbers, silicone rubbers and silasticpolymers, polyesters, polyimides, cellulose and cellulose derivatives(such as cellulose acetate, nitrocellulose and the like), acrylates,methacrylates, vinyl polymers (such as polyvinyl acetate, polyvinylchloride, polyvinylidene chloride, polyvinyl fluoride, and the like),polystyrene and styrene graft copolymers, styrene-acrylonitrilecopolymers, rayon, nylon, polyvinylbutyrate, polyformaldehyde, etc.Other materials which may be employed as the surface are silica gel,silicon wafers, glass, paper, insoluble protein, metals, metalloids,metal oxides, magnetic materials, semiconductive materials, cements orthe like. In addition are included substances that form gels, i.e.,proteins such as gelatins, lipopolysaccharides, silicates, agarose,polyacrylamides or polymers which form several aqueous phases such asdextrans, polyalkylene glycols (alkylene with 2 to 3 carbon atoms) orsurfactants, e.g. amphophilic compounds such as phospholipids, longchain (12-24 carbon atoms) alkyl ammonium salts and the like.

[0063] The surface may comprise polystyrene, styrene copolymersincluding styrene(vinyl monomer) copolymers such asstyrene-acrylonitrile copolymers, polyolefins such as polyethylene andpolypropylene, and acrylate and methacrylate polymers and copolymers andmixtures thereof.

[0064] The surface may also comprise magnetizable materials inparticulate form. Traditional interference by such magnetizablematerials may be minimized by adding magnetizable particles to each ofthe reaction steps. Magnetic interference produced at each step may bemade nearly equal, and thus is effectively cancelled. Magnetizableparticles may be easily separated from the serum or other solution byapplication of a magnetic field to concentrate the particles.

[0065] The capture antibody may be bound to the surface by any method ofbonding which does not significantly reduce the antibody binding sitesand which binds sufficiently to permit separation of the surface fromthe liquids and rinse solutions without significant detachment ofantibody from the surface. Non-covalent bonding may be achieved byadsorption, ionic bonding, van der Waals adsorption, electrostaticbonding, and other non-covalent bonding. The antibody may also be boundto the surface by covalent bonding.

[0066] Procedures for covalently adhering antibodies to surfaces aredescribed by I. Chibata in IMMOBILIZED ENZYMES, Halsted Press, New York,1978, and by A. Cuatrecasas, J.Bio.Chem. 245:3059 (1970), the entirecontents of which are hereby incorporated by reference. The surface maybe coated with a protein and coupled with antibody using the proceduresdescribed in U.S. Pat. No. 4,210,418 using glutaraldehyde as a couplingagent, for example. In an alternate procedure, the surface may be coatedwith a layer having free isocyanate groups such as a polyetherisocyanate. Application of the antibody in aqueous solution theretoeffects the requisite bonding. In another procedure, the antibody may becoupled to a hydroxylated material by means of cyanogen bromide asdescribed in U.S. Pat. No. 3,720,760. In a still further procedure,Staphylococcus Protein A may be bound to the surface, and the F_(c)chain of the antibody can be conjugated with the Protein A.

[0067] Capture antibodies may be attached to a surface by adhesionfollowed by chemical crosslinking. Crosslinking agents which may be usedinclude, but are not limited to,1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), glutaraldehyde,adipic acid dihydrazide, bis-diazotized benzidine, 1,4-butane diglycidylether, bis-maleimido hexane, sulfosuccinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate, andN-hydroxysuccinimidyl 4-azidosalicylic acid. EDC may be used for anysurface that has free carboxyl groups. These and many other similarreagents are well known in the art.

[0068] 6. Conjugation of Antibodies to a Common Member

[0069] In another exemplary embodiment, the same or different antibodiesmay be conjugated to a common member, using any of the conjugationmethods described above. Common members include, but are not limited to,a particle, microparticle (FIG. 6B), polypeptide (FIG. 6C), chemicallinker, or a surface (FIG. 6A) as described above.

[0070] A common member may be conjugated with more than one antibody,each antibody binding to a different epitope on the analyte beingassayed. See FIG. 6. The multiple antibodies may be conjugated to thecommon member at controlled molar ratios, for example using methodswhere the stoichiometry of the conjugation reaction is controlled.

[0071] In addition, multiple common members may be individuallyconjugated with a different antibody, each antibody binding to adifferent epitope on the analyte being assayed. See FIG. 7. Molar ratiosof the different antibodies may be controlled, using methods such as themixing of multiple common members with conjugated antibodies.

[0072] a. Microparticle

[0073] Antibodies may be attached to microparticles using any of theconjugation methods described above. Various factors may be consideredwhen choosing the coupling method including, but not limited to type andsize of particle, coupling reagents, concentration of reactants, singleor multi-step reaction, reaction buffer and pH, storage buffer, andblocking agents. For more information on using microparticles, see BangsTechNote #201 “Working with Microspheres”; Bangs TechNote #204“Adsorption to Microspheres”; Bangs TechNote #205 “Covalent Coupling”;Seradyn Technical Method Bulletin “Recommended Adsorption and CovalentCoupling Procedure” (1999); Hager, H. J. “Latex Polymer Reagents forDiagnostic Tests”; U.S. Pat. No. 3,857,931; and Wong, Chemistry ofProtein Conjugation and Cross-Linking, 1991, CRC Press, Boca Raton,Fla., which are incorporated herein by reference.

[0074] 7. Signal

[0075] Signal reagents may be prepared by conjugating an antibody to asignal-generating element by any of a number of common methods.Conjugates may be prepared, for example, by utilizing free sulfhydrylgroups, generating sulflhydryl groups from available disulfide bonds, orintroducing additional sulfhydryls onto the antibody. Linking agents,such as succinimydyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(SMCC) may then used to conjugate the activated antibody to thesignal-generating element. For more information on preparing antibodyconjugates, see Pierce Instructions “ImmunoPure IgG1 Fab and F(ab′)2Preparation Kit” #44880; Pierce Instructions “SMCC, Sulfo-SMCC” #22360,#22322; Pierce Instructions “EZ-Link Maleimide Activated PhosphataseKit” # 31493; Beale, D. (1987) Molecular fragmentation: Someapplications in immunology, Exp Comp Immunology 11, 287-296; Lamoyi, E.(1986) Preparation of F(ab′)2 fragments from mouse IgG of varioussubclasses, Meth Enz 121, 652-663; King, T P, Kochoumian, L. (1979), Acomparison of different enzyme-antibody conjugates for enzyme-linkedimmunosorbent assay, J Immun Meth 28, 201-210; Brinklay, Mass. (1992), Asurvey of methods for preparing protein conjugates with dyes, haptensand crosslinking reagents, Bioconj Chem 3, 2-13; and TechNote #204,“Adsorption to Microspheres”, Bangs Laboratories Inc Rev. #001 Aug. 4,1999, which are incorporated herein by reference.

[0076] Signal-generating elements of the present invention include, butare not limited to, radiolabels, metal particles, chromogens,fluorescent dyes, labeled proteins and enzymes. When thesignal-generating element is an enzyme, the enzyme may catalyze areaction wherein the depletion of substrate or the production of productcan be detected. Exemplary enzymes include, but are not limited to,amylases, polynucleotidase, arginase, adenase, aminopolypeptidase,pepsin, lipases, catalase, tyrosinases, alcohol dehydrogenase, succinicdehydrogenase, diaphorase, glyoxalase, aldolase, glucose oxidase,horseradish peroxidase, beta-galactosidase, phosphatases, phosphorylasesand hexokinases. Exemplary enzymes also include alkaline phosphatase,glucose oxidase, horseradish peroxidase, β-galactosidase and phenoloxidase. The enzymatic conversion of a substrate to a product may bemeasured by for example optical and electrochemical means.

[0077] a. Multiple Antibodies Conjugated to a Signal-Generating Element

[0078] The sensitivity of a sandwich assay is limited largely by thelevel of non-specific adsorption of the signal reagent to the capturereagent and surfaces within the detection region of the analysis device.Such surfaces may include the walls of a cuvette, a wicking element, anelectrode and the like. When the analyte is at low levels in a sample, asignificant amount of the signal measured is background, and thus notrelated to the concentration of the analyte in the sample. Thepropensity of the signal reagent to non-specifically adsorb to surfaceswithin the analysis device may be a function of the inherent propertiesof the antibody and signal-generating element used to form the signalantibody, as well as the crosslinker. Additionally, the materials usedin the analysis device (e.g., types of plastics) and the composition ofthe wash and substrate-containing solutions are generally optimized tominimize such non-specific protein adsorption. In particular, varioussurfactants, e.g. Tween 20, Brij 35, Triton X100, are almost invariablyused in sandwich assays to minimize unwanted interactions of the signalreagents with other components within the assay device. Non-enzymaticproteins such as serum albumin, denatured proteins such as gelatins, anddeactivated enzymes may also be added as surface blocking agents inorder to lower the level of non-specific adsorption of the signalreagent. Even when all components of the assay are optimized, there isstill usually a detectable level of non-specific signal reagentadsorption. The level of this non-specific adsorption is proportional tothe amount of signal reagent used in the assay. Therefore, using lesssignal reagent or using the same molar amount of a lower molecularweight signal reagent will usually result in a lower background signalin the assay.

[0079] The signal reagent may comprise fragments of antibodies, such asFab fragments, which may contribute to lowering the level of backgroundsignal. By lowering the molecular weight of the signal reagent, the timerequired for the binding step may be reduced due to diffusionalconsiderations. The signal reagent may also comprise more than oneantibody, which may also lower background signal levels by minimizingthe overall size of the signal reagent while maximizing its ability togenerate a signal in response to the presence of the analyte ofinterest. The more than one antibody of the signal reagent may bind totwo or more different epitopes of the analyte, which may lead tostabilization of the signal antibody-analyte complex when more than oneepitope of the analyte is bound by more than one antibody of the signalreagent.

[0080] When the signal reagent comprises more than one antibody, saidantibodies may bind to one or more different epitopes.

[0081] A signal-generating element may be conjugated with more than oneantibody, each antibody binding to a different epitope on the analyte ofinterest. See FIG. 6C. The molar ratio of antibodies conjugated to thesignal-generating element may be controlled by selecting the reactionconditions and stoichiometry for the conjugation reaction.

[0082] A first antibody may be conjugated to a first signal-generatingelement and a second antibody may be conjugated to a secondsignal-generating element, each at a ratio greater than 1:1, and thenmixed, wherein the first and second antibodies each bind to a differentepitope on the analyte of interest. See FIG. 7B. The molar ratio of thefirst and second antibodies may also be controlled.

[0083] The signal-generating element may be conjugated to a singleantibody, or two or more antibodies to different epitopes on the sameanalyte, wherein the signal antibody comprises a ratio ofsignal-generating element to a population of antibodies from about 1:100to about 1:1.001. The signal-generating element may also be conjugatedto a population of antibodies at a ratio from about 1:90 to about1:1.001. The signal-generating element may also be conjugated to apopulation of antibodies at a ratio from about 1:80 to about 1:1.001.The signal-generating element may also be conjugated to a population ofantibodies at a ratio from about 1:70 to about 1:1.001. Thesignal-generating element may also be conjugated to a population ofantibodies at a ratio from about 1:60 to about 1:1.001. Thesignal-generating element may also be conjugated to a population ofantibodies at a ratio from about 1:50 to about 1:1.001. Thesignal-generating element may also be conjugated to a population ofantibodies at a ratio from about 1:40 to about 1:1.001. Thesignal-generating element may also be conjugated to a population ofantibodies at a ratio from about 1:30 to about 1:1.001. Thesignal-generating element may also be conjugated to a population ofantibodies at a ratio from about 1:20 to about 1:1.001. Thesignal-generating element may also be conjugated to a population ofantibodies at a ratio from about 1:10 to about 1:1.001, about 1:20 toabout 1:10, about 1:30 to about 1:20, about 1:40 to about 1:30, about1:50 to about 1:40, about 1:60 to about 1:50, about 1:70 to about 1:60,about 1:80 to about 1:70, about 1:90 to about 1:80, and about 1:100 toabout 1:90. The signal-generating element may also be conjugated to apopulation of antibodies at a ratio from about 1:5 to about 1:1.001,about 1:10 to about 1:5, about 1:15 to about 1:10, about 1:20 to about1:15, about 1:25 to about 1:20, about 1:30 to about 1:25, about 1:35 toabout 1:30, about 1:40 to about 1:35, about 1:45 to about 1:40, andabout 1:50 to about 1:45. The signal-generating element may also beconjugated to a population of antibodies at a ratio of about 1:3.3.

[0084] The signal-generating element may be conjugated to a first andsecond antibody, each capable of binding to different epitopes on ananalyte or subforms thereof, wherein the ratio of signal-generatingelement to total antibody is as disclosed above, and wherein the ratioof the first antibody to the second antibody is from about 1:50 to about50:1. The ration of the first antibody to the second antibody may alsobe from about 1:10 to about 10:1, from about 1:5 to about 5:1, fromabout 1:3 to about 3:1, and from about 1:2 to about 2:1.

[0085] 8. Signal and Detection

[0086] A signal may be produced by the signal-generating element, whichmay be measurable by a sensing element using methods which include, butare not limited to, radiation, optically, electrochemically, or by someother transduction means known in the art. A sensing element may be anelectrode, biosensor, field-effect transistor, surface acoustic wavedevice, optical wave-guide, fiber optic, cuvette, radioactivitydetector, reagents that facilitate, physical, nuclear, chemical,biochemical, electrical, or optical detection, or combinations thereof.

[0087] 9. Immunoassay Device

[0088] The immunoassay of an analyte of interest may be conducted in animmunoassay device comprising one or more sensing elements and a surfaceon which a modified sandwich immunoassay is conducted as describedabove. The device surface may comprise two or more antibodies that areeach capable of binding to different epitopes on the analyte. At leastone of the two antibodies is also capable of binding to the same epitopeon a subform of the analyte. At least one of the epitopes on the analyteis unavailable for binding on the subform.

[0089] Devices of the present invention include a cartridge, columns,syringe, cuvette, or other analytical device or system known in the art.The cartridge may be of a type as described in U.S. Pat. No. 5,096,669.Examples of other cartridge configurations are found in U.S. Pat. Nos.5,416,026, 5,593,638, 5,447,440, 5,628,961, 5,514,253, 5,609,824,5,605,664, 5,614,416, 5,789,253, 6,030,827, and 6,379,883. Still othercartridge configurations are described in PCT/US00/31158, PCT/US01/04345and copending U.S. patent application Ser. No. 10/087,730. Thedisclosures of the foregoing are hereby incorporated by reference.

[0090] The surface of the immunoassay device may be of any appropriatesurface known to those of skill in the art including, but not limitedto, glass, semiconductor, plastic, silicon dioxide, photoformable PVA,photoformable gelatin, film forming latex, and conductive metal. Aconductive metal surface may be silver, iridium, gold, platinum, andalloys thereof.

[0091] Antibodies may be absorbed, adsorbed or covalently attached tothe surface of the immunoassay device. Antibodies may be absorbed intoPVA, gelatin and latex layers or absorbed to their surfaces. Antibodiesmay be adsorbed to glass, metal, semiconductor, plastic, silicondioxide, photoformable PVA, and conductive metal. The two or moreantibodies may also be attached to a microparticle as discussed above,wherein the microparticle is attached to the surface as discussed above.The immunoassay device may also contain a third antibody which binds toa different epitope on the analyte.

[0092] The sensing element of the immunoassay device may be of any typeknown in the art including, but not limited to, an electrode, biosensor,field-effect transistor, surface acoustic wave device, opticalwave-guide, fiber optic, cuvette, radioactivity detector,immunochromatographic device, reagents that facilitate, physical,nuclear, chemical, biochemical, electrical, optical detection, orcombinations thereof.

[0093] 10. Immunoassay Kit

[0094] An immunoassay kit may be used to detect an analyte of interestand subforms thereof in a modified sandwich immunoassay as describedabove. The kit may comprise three or more antibodies that are eachcapable of binding to different epitopes on the analyte. At least two ofthe three antibodies are also capable of binding to the same epitopes ona subform of the analyte. At least one of the epitopes on the analyte isunavailable for binding on the subform.

[0095] 11. Sandwich Immunoassay Product

[0096] A sandwich immunoassay product may comprise an analyte ofinterest and a subform thereof. At least three epitopes on the analyteare available for binding by at least three different antibodies. Theanalyte may be bound by at least three different antibodies with eachantibody binding to a different epitope on the analyte. At least two ofthe three epitopes on the analyte are available for binding on thesubform by at least two of the at least three different antibodies. Thesubform may be bound by at least two of the three different antibodiesthat bind to the analyte with each antibody binding to a differentepitope on the subform. At least one epitope of the analyte isunavailable for binding on the subform. At least one of the antibodiesbound to the analyte and the subform of the analyte may be a signalantibody.

[0097] 12. Method of Assaying for an Analyte

[0098] A sample may be immunoassayed for the presence of an analyte andsubforms thereof by performing a modified sandwich immunoassay asdescribed above. At least three different antibodies are used, whereineach antibody is capable of binding to a different epitope on theanalyte. At least two of the antibodies capable of binding to theanalyte are capture antibodies or signal antibodies, or a combinationthereof. At least one of the epitopes on the analyte is unavailable forbinding on the subform.

[0099] The sample to be immunoassayed is added to a surface comprisingone or more capture antibodies. The one or more signal antibodies arethen added. Alternatively, one or more signal antibodies may be added tothe sample before or during application of the sample to the surfacecomprising one or more capture antibodies. After removing unbound signalantibodies, the extent of binding of the one or more signal antibodiesis determined as described above. A signal will be produced by analyteas well as all subforms of the analyte that have at least one epitopethat is present or capable of being bound by a capture antibody and atleast a second epitope that is present or capable of being bound by asignal antibody.

[0100] 13. Method of Diagnosing an Acute Disease

[0101] The analyte of interest and subforms thereof may be immunoassayedto determine whether a patient has suffered an acute medical event. Asample may be obtained from the patient and immunoassayed for thepresence of the analyte of interest and subforms thereof by using themethod of assaying described above. Signal produced by signal antibodiesmay be compared to control values. A signal, or a sequence of signalsfrom a series of assays over a period of hours or days, detectable abovecontrol may indicate that the patient has suffered an acute medicalevent.

[0102] The acute medical event may be any disease state that isassociated with a clinical marker, wherein the clinical marker is ananalyte in the modified immunoassay described herein. The medical eventmay be for example a myocardial infarction. The analyte of interest maybe for example TnI, TnT, TnC, CK-M, CK-B, CK-MB, myoglobin, TSH, FSH,CRP, BNP, pro-BNP, PSA, PCA, apolipoprotein, and combinations thereof.

[0103] 14. Method of Diagnosing the Time of the Occurrence of an AcuteDisease

[0104] The analyte of interest and subforms thereof may be immunoassayedto determine when a patient suffered an acute medical event. A sample,or set of samples obtained at different times, may be obtained from thepatient and immunoassayed for the presence of the analyte of interestand subforms thereof by using the method of assaying described above.The amount of signal produced by signal antibodies may be correlatedwith a standard curve of amount of analyte vs. time to determine whenthe patient suffered the acute medical event. One of ordinary skill mayproduce the standard curve by using the methods described herein.

[0105] 15. Method of Diagnosing the Severity of an Acute Disease

[0106] The analyte of interest and subforms thereof may be immunoassayedto determine the severity of an acute medical event suffered by apatient. A sample, or set of samples obtained at different times, may beobtained from the patient and immunoassayed for the presence of theanalyte of interest and subforms thereof by using the method of assayingdescribed above. The amount of signal produced by signal antibodies maycorrelate to the severity of a medical event suffered by the patient.

[0107] Having now generally described the invention, the followingexamples are provided in order to more fully illustrate the invention.These examples are for purposes of illustration only and are notintended to limit the scope of the invention.

EXAMPLE 1

[0108] Conjugation of cTnI Capture Antibodies to a Particle

[0109] Bead particles conjugated with antibodies specific for cTnI wereprepared by first exchanging polystyrene/acrylic acid latex 0.2 μmdiameter spheres (Seradyn) into 0.05 M 2-(N-morpholino)ethanesulfonicacid (MES, Sigma Aldrich) buffer at pH 6.2. To a 2% w/w solution of thelatex spheres was added cTnI antibodies (buffer exchanged into the same0.05 M MES buffer) at a controlled mass ratio relative to the mass ofthe latex spheres. The solution was stirred for 15 minutes at 4° C. andthe antibody coated beads separated via controlled centrifugation.Typically sufficient antibody was added to fully saturate the latex beadsurface (8% to 15% of the mass of the latex particles). Themicroparticle centrifuged pellet was then resuspended into MES buffer at1% w/v density and 1 to 10 mM of EDC was added to covalently crosslinkthe adsorbed antibody to the latex spheres. The suspension was allowedto react for 4 to 12 hours at which time the latex particles wereseparated by centrifugation, resuspended at 1% in the MES buffer and theEDC addition was repeated. Using the above method, microparticle capturereagents were made comprising either CB1 (19C7, Hytest Inc.) or CB2(34503228P, Biospacific Inc.). CB12 microparticles comprising both CB1and CB2 were prepared using the above method by adding a mixture of thetwo capture antibodies.

EXAMPLE 2

[0110] Preparation of Signal Antibodies

[0111] The following method was used to prepare alkaline phosphatase(ALP) labeled Fab conjugates to be used as signal reagents. Alkalinephosphatase (Biozyme) was buffer exchanged into phosphate bufferedsaline (PBS). A 1 mg/ml to 15 mg/ml solution of the ALP was reacted with25 weight percent (relative to the ALP weight in the solution) of theheterobifunctional crosslinkersuccinimidyl-4-[N-maleimidomethyl]-cyclohexane-1-carboxy[6-amidocaproate](LCSMCC, Pierce). The crosslinker was added as a solution in dimethylsulphoxide (DMSO, Sigma). This solution was allowed to react at roomtemperature for 45 minutes, after which it was centrifuged to removeprecipitates. The activated ALP was separated from unreacted LCSMCC viaa Sephadex G50 desalting column (Sigma Aldrich).

[0112] Fab fragments of the antibodies to be incorporated into thesignal reagent were formed via pepsin (Sigma) digestion of the wholeantibodies and separated using a S200 sephacryl size exclusion column(Amersham Pharmacia). The resulting F(ab)₂ fragments were chemicallyreduced in 60 mM mercaptoethylamine (MEA, Sigma Aldrich) for 45 minutesat 37° C. to produce two Fab fragments, which were separated from excessMEA by desalting using a sephadex G50 column. The Fab fragments werethen added to activated ALP and allowed to react at 4° C. for 2 to 12hours. The reaction was quenched with an equal volume of 0.1 M glycine(Sigma), 0.01 M cysteine (Pierce) in PBS pH 7.4. The reaction productswere then fractionated on a Sephacryl S200 column to yield the signalreagent comprising Fab fragments. Signal reagents with specific molarratios of Fab fragments to ALP were obtained by varying the ratio ofFab₂ fragments to ALP in the reaction above. FIG. 12 shows the elutionprofile for ALP-Fab signal reagents with ALP:Fab ratios of 1:1, 1:3, 1:6and 1:10.

[0113] Using the above method, ALP-labeled signal reagents were madecomprising either EC1 (G129C, Biospacific Inc.) or EC2 (G130C,Biospacific Inc.). EC12 signal reagents comprising both EC1 and EC2 wereprepared using the above method by mixing the two fab fragments at amolar ratio of 1:1 before adding the mixture to activated ALP

EXAMPLE 3

[0114] Preparation Of An ELISA Sensor With cTnI Capture Antibodies

[0115] The following method was used to prepare an analyte detectorcomprising an immobilized layer of antibody labeled particles on anELISA sensor. Briefly, amperometric sensors used in the analyte detectorare fabricated using traditional semiconductor thin layer techniques onoxidized silicon wafers. The cTnI capture regions are produced bymicrodispensing solutions of the capture antibodies described in Example1 using methods described in U.S. Pat. No. 5,554,339. Droplets of theparticle-containing suspension are allowed to dry to form cTnI captureregions on the sensor surface. The sensor chips are assembled with aseparated electrochemical grounding chip into a plastic base coveredwith a two sided adhesive tape gasket, laser cut with openings for thesensors, ground chip and fluidic channels. To this base assembly aplastic cover is added to form fluidic conduits. The enzyme labeledsignal reagent of Example 2 is printed onto the same ELISA sensor as thecapture antibodies. The signal reagent is formulated in a 1% to 50%sugar solution containing PBS and a preservative. This composition wasfound to provide rapid dissolution of the signal reagent into a bloodsample. The assembled cartridge is pressed to yield the disposablecartridge, which may be used in a portable electrochemical analyzer.

EXAMPLE 4

[0116] Comparison Of Detection Of cTnI Using Single or Multiple CaptureAntibodies

[0117] An analytical system for measuring cTnI was used of the generaltype described in U.S. Pat. No. 5,096,669, which is hereby incorporatedby reference. Briefly, cartridges for assaying cTnI were produced usingsensors coated with latex microsphere comprising CB1, CB2 and CB12capture reagents, as described in Example 3. cTnI was assayed in threewhole-blood samples to which either free cTnI (Scripps Laboratories) orITC complex (Hytest Ltd) was added. The signal reagent EC12, asdescribed in Example 2, was used to produce an amperometric signal basedon dephosporylation of a substrate by ALP to produce an electroactiveproduct. The amount of electroactive product is proportional to theamount of detectable cTnI.

[0118]FIG. 10 shows high signal levels for cartridges comprising the CB1capture reagent for both free cTnI (diamond points) and the two levelsof ITC complex (square and triangle points). By contrast, cartridgescomprising the CB2 capture reagent yield lower signals, especially forthe ITC complex. Cartridges using the CB12 hybrid capture reagent showimproved signal generation for both forms of cTnI relative to either CB1or CB2 capture reagents. The improved performance of the CB12 capturereagent is surprising, because the hybrid reagent comprises the CB2antibody that by itself has markedly lower analytical performance thanthe CB1 antibody.

EXAMPLE 5

[0119] Comparison of Detection of cTnI Using Single or Multiple SignalAntibodies

[0120] cTnI was assayed in a whole blood sample spiked with a 6.0 ng/mLcITC complex immediately after preparation and after a one-dayincubation at room temperature using cartridges with sensors coated withlatex microsphere comprising CB1 and CB12 capture reagents, as describedin Example 3, and EC1, EC2 or EC12 signal reagents as described inExample 2. FIG. 11 demonstrates that the EC1 signal reagent shows ahigher signal generation than the EC2 signal reagent. After a one dayincubation of the sample to simulate aging of cTnI (e.g., proteolyticdegradation), the signal level for the EC1 signal reagent dropssignificantly. In contrast, the EC2 signal reagent show only a modestdrop in the signal level. The signal generation of the hybrid signalreagent EC12 is surprisingly superior to each of the two single antibodysignal reagents, especially after incubating the sample for one day.

EXAMPLE 6

[0121] Detection of cTnI in Serum as a Diagnosis of MyocardialInfarction

[0122] Whole blood samples are obtained from patients suspected ofsuffering a myocardial infarction, as well as from control individuals.cTnI is assayed in the whole blood samples using cartridges with sensorscoated with latex microsphere comprising CB12 capture reagents, asdescribed in Example 3, and EC12 signal reagents as described in Example2. The signal generated in samples derived from patients suspected ofsuffering an MI is compared to the level of signal from controls. Levelsof signal greater than control indicate that the patient has suffered aMI.

What is claimed is:
 1. An immunoassay composition for detecting ananalyte comprising at least three different antibodies, the at leastthree different antibodies being capable of binding to at least threedifferent epitopes on an analyte, in which at least two of the at leastthree different antibodies are capable of binding to at least twodifferent epitopes on a subform of the analyte, and in which at leastone of the at least three different epitopes on the analyte isunavailable for binding on the subform.
 2. An immunoassay compositionfor detecting an analyte comprising m different antibodies, (a) in whichat least n of the m different antibodies are capable of binding to ndifferent epitopes on an analyte, and (b) in which no more than n−1 ofthe n different epitopes on the analyte are available for binding on asubform of the analyte, provided that m and n are greater than or equalto 3, and m is greater than or equal to n.
 3. The immunoassaycomposition of claim 1 in which the analyte comprises TnI, TnT, TnC,CK-M, CK-B, CK-MB, myoglobin, TSH, FSH, CRP, BNP, pro-BNP, PSA, PCA,apolipoprotein, or combinations thereof.
 4. The immunoassay compositionof claim 1 in which at least one of the at least three antibodiescomprises a full length antibody, a single-chain antibody, or anantibody fragment.
 5. The immunoassay composition of claim 4 in whichthe antibody fragment comprises a Fab fragment.
 6. The immunoassaycomposition of claim 1 in which the analyte is cTnI.
 7. The immunoassaycomposition of claim 1 which further comprises a common memberconjugated to at least one of the at least three antibodies.
 8. Theimmunoassay composition of claim 7 in which the at least one of the atleast three antibodies is covalently attached to the common member. 9.The immunoassay composition of claim 8 in which the covalent attachmentcomprises a crosslinker, which is derived from a crosslinking agent. 10.The immunoassay composition of claim 9 in which the crosslinking agentcomprisessuccinimidyl-4-[N-maleimidomethyl]-cyclohexane-1-carboxy[6-amidocaproate],glutaraldehyde, adipic acid dihydrazide, bis-diazotized benzidine,1,4-butane diglycidyl ether, bis-maleimido hexane, sulfosuccinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate, orN-hydroxysuccinimidyl 4-azidosalicylic acid.
 11. The immunoassaycomposition of claim 7 in which the common member is a microparticle.12. The immunoassay composition of claim 11 in which the microparticlecomprises latex beads, polystyrene beads, polystyrene/acrylic acidbeads, or combinations thereof.
 13. The immunoassay composition of claim7 in which the common member comprises a signal-generating element. 14.The immunoassay composition of claim 13 in which the signal-generatingelement comprises a radiolabel, metal particle, fluorescent dye,chromogenic dye, labeled protein, enzyme, or combinations thereof. 15.The immunoassay composition of claim 14 in which the enzyme comprisesperoxidase, glucose oxidase, phenol oxidase, β-galactosidase, alkalinephosphatase, or combinations thereof.
 16. The immunoassay composition ofclaim 13 in which the molar ratio of the at least one of the at leastthree antibodies conjugated to the signal-generating element is fromabout 1:1 to about 10:1.
 17. The immunoassay composition of claim 16 inwhich the molar ratio of the at least one of the at least threeantibodies conjugated to the signal-generating element is about 3:1. 18.The immunoassay composition of claim 13 in which at least two of the atleast three antibodies are conjugated to the signal-generating element.19. The immunoassay composition of claim 18, in which the molar ratio ofthe at least two of the at least three antibodies conjugated to thesignal-generating element is from 1:1 to 10:1.
 20. An immunoassaycomposition for detecting an analyte comprising four differentantibodies, the four different antibodies being capable of binding tofour different epitopes on an analyte, in which at least one of fourdifferent epitopes on the analyte is unavailable for binding on asubform of the analyte, in which a first and second of the fourdifferent antibodies are conjugated to a surface, in which a third andfourth of the four different antibodies are each conjugated to anenzyme, in which at least one of the first and second antibodies iscapable of binding to at least one epitope on the subform of theanalyte, and in which at least one of the third and fourth antibodies iscapable of binding to at least one epitope on the subform.
 21. Animmunoassay composition for detecting an analyte comprising m differentantibodies, (a) in which n of the m different antibodies are capable ofbinding to n different epitopes on an analyte, (b) in which no more thann−1 of the n different epitopes on the analyte are available for bindingon a subform of the analyte, (c) in which p of the n differentantibodies are conjugated to a surface, (d) in which p of the ndifferent antibodies are each conjugated to an enzyme, (e) in which atleast p−1 of the p antibodies in step (c) is capable of binding to atleast p−1 different epitopes on the subform of the analyte, and (f) inwhich at least p−1 of the p antibodies in step (d), is capable ofbinding to at least p−1 different epitopes on the subform of theanalyte. provided that m and n are 4, and that p is
 2. 22. Animmunoassay device comprising one or more sensing elements and at leastone surface on which an immunoassay can be conducted for detecting ananalyte, which at least one surface comprises at least two differentantibodies, the at least two different antibodies being capable ofbinding to at least two different epitopes on the analyte, in which atleast one of the at least two different antibodies is capable of bindingto at least one epitope on a subform of the analyte, in which at leastone of the at least two different epitopes on the analyte is unavailablefor binding on the subform, and in which the at least two differentantibodies are bound to the at least one surface.
 23. An immunoassaydevice comprising one or more sensing elements and at least one surfaceon which an immunoassay can be conducted for detecting an analyte, (a)in which at least one surface comprises m different antibodies, (b) inwhich at least n of the m different antibodies are capable of binding ton different epitopes on the analyte (c) in which no more than n−1 of then different epitopes on the analyte are available for binding on asubform of the analyte, and (d) in which at least n of the m differentantibodies are bound to the at least one surface, provided that m and nare greater than or equal to 2, and m is greater than or equal to n. 24.The immunoassay device of claim 21(a) in which the analyte comprisesTnI, TnT, TnC, CK-M, CK-B, CK-MB, myoglobin, TSH, FSH, CRP, BNP,pro-BNP, PSA, PCA, apolipoprotein, or combinations thereof.
 25. Theimmunoassay device of claim 21(a) in which the at least one surfacecomprises glass, semiconductor, plastic, silicon dioxide, photoformablePVA, photoformable gelatin, film forming latex, conductive metal, orcombinations thereof.
 26. The immunoassay device of claim 25 in whichthe conductive metal comprises silver, iridium, gold, platinum, orcombinations thereof.
 27. The immunoassay device of claim 21(a) in whichat least one of the at least two antibodies is absorbed or adsorbed tothe at least one surface.
 28. The immunoassay device of claim 21(a) inwhich at least one of the at least two antibodies is covalently attachedto the at least one surface.
 29. The immunoassay device of claim 21(a)which further comprises a microparticle in which the at least twoantibodies are bound to the microparticle and in which the microparticleis bound to the at least one surface.
 30. The immunoassay device ofclaim 21(a) which further comprises at least two microparticles in whichonly one of the at least two antibodies is bound to one of the at leasttwo microparticles and in which the at least two microparticles arebound to the at least one surface.
 31. The immunoassay device of claim21(a) which further comprises a third antibody which binds to adifferent epitope on the analyte and the subform of the analyte.
 32. Theimmunoassay device of claim 21(a) in which the sensing element comprisesan electrode, biosensor, field-effect transistor, surface acoustic wavedevice, optical wave-guide, fiber optic, cuvette, radioactivitydetector, immunochromatographic device, reagents that facilitatephysical, nuclear, chemical, biochemical, electrical, or opticaldetection, or combinations thereof.
 33. An immunoassay kit comprising ina suitable container, at least three different antibodies, the at leastthree antibodies being capable of binding to at least three differentepitopes on an analyte, in which at least two of the at least threedifferent antibodies are capable of binding to at least two differentepitopes on a subform of the analyte, and in which at least one of theat least three different epitopes on the analyte is unavailable forbinding on the subform.
 34. An immunoassay kit comprising in a suitablecontainer m different antibodies, (a) in which at least n of the mdifferent antibodies are capable of binding to n different epitopes onan analyte, and (b) in which no more than n−1 of the n differentepitopes on the analyte are available for binding on a subform of theanalyte, provided that m and n are greater than or equal to 3, and m isgreater than or equal to n.
 35. The immunoassay kit of claim 33 in whichthe analyte comprises TnI, TnT, TnC, CK-M, CK-B, CK-MB, myoglobin, TSH,FSH, CRP, BNP, pro-BNP, PSA, PCA, apolipoprotein, or combinationsthereof.
 36. A sandwich immunoassay product comprising an analyte and asubform of the analyte, in which at least three different epitopes onthe analyte are available for binding by at least three differentantibodies, in which at least two of the three different antibodies arebound to a different epitope on the analyte, in which two of the threedifferent epitopes on the analyte are available for binding on thesubform, in which at least two of the three different antibodies arebound to a different epitope on the subform, and in which at least oneof the at least three different epitopes on the analyte is unavailablefor binding on the subform.
 37. A sandwich immunoassay productcomprising an analyte, a subform of the analyte, and at least 2 of mdifferent antibodies, (a) in which the analyte includes at least ndifferent epitopes each capable of being bound by one of the m differentantibodies, (b) in which at least 2 of the m different antibodies areeach bound to a different epitope on the analyte, (c) in which no morethan n−1 of the n different epitopes on the analyte are available forbinding on a subform of the analyte, and (d) in which at least 2 of them different antibodies are each bound to a different epitope on thesubform of the analyte, provided that m and n are greater than or equalto 3, and that m can be greater than or equal to n.
 38. The sandwichimmunoassay product of claim 36 in which the analyte comprises TnI, TnT,TnC, CK-M, CK-B, CK-MB, myoglobin, TSH, FSH, CRP, BNP, pro-BNP, PSA,PCA, apolipoprotein, or combinations thereof.
 39. The sandwichimmunoassay product of claim 36 in which at least one of the at leastthree antibodies and least one of the at least two antibodies comprisesa signal-generating element.
 40. A method of determining whether apatient has suffered a myocardial infarction comprising: (a) applying asample from a patient suspected of suffering a myocardial infarction toa surface to which is bound at least two antibodies, in which the atleast two antibodies are capable of binding to at least two differentepitopes on cTnI, in which at least one of the at least two antibodiesis capable of binding to at least one different epitope on a subform ofcTnI, and in which at least one epitope of cTnI is unavailable forbinding on the subform; (b) adding a reagent comprising a third antibodywhich binds to yet another epitope on cTnI and the subform; and (c)determining the extent of binding of the third antibody.
 41. A method ofdetermining whether a patient has suffered a myocardial infarctioncomprising: (a) applying a sample from a patient suspected of sufferinga myocardial infarction to a surface to which is bound a first antibodywhich is capable of binding to a first epitope on cTnI and a subform ofcTnI; (b) adding separately or together at least a second and thirdantibody, in which the at least second and third antibodies are capableof binding to at least two different epitopes on cTnI, in which the atleast second and third antibodies are not capable of binding to thefirst epitope, in which at least one of the at least second and thirdantibodies are each capable of binding to at least one different epitopeon the subform of cTnI, and in which at least one epitope of cTnI isunavailable for binding on the subform; and (c) determining the extentof binding of the at least second and third antibodies.